1. Field of the Invention
The present invention generally relates to improved methods of nuclear transfer, permitting efficient development of cybrids developed using male, as well as female, donor cells. The present invention provides for the transfer of nuclei of long-term cultured, non-fetal, somatic cells into enucleated oocytes to produce viable totipotent cybrids capable of generating into an embryo, fetus, and/or animal. The present invention further provides for targeted genetic manipulation of the genome of the donor cell to produce a desired genetically-altered animal.
2. Background of the Invention
Recent discoveries in animal cloning have led to a new revolution in science. There is no longer any doubt of the potential applications of cloning technologies in agriculture, medicine and basic biological research. Cloning offers an inexpensive and more effective way to produce transgenic animals than conventional microinjection procedures.
Methods for cloning animals, in particular mammals, have been sought and developed in earnest over the past two decades. A predominant technique used today for cloning is known as “nuclear transfer” or “nuclear transplantation”. Nuclear transfer procedures are well known in the art and are described in many references (See, e.g., Campbell et al., Theriogenology, 43: 181 (1995); Collas et al., Mol. Report Dev., 38: 264–267 (1994); Keefer et al., Biol. Reprod., 50: 935–939 (1994); Sims et al., Proc. Natl. Acad. Sci., USA, 90: 6143–6147 (1993); WO 97/07668; WO 97/07669; WO 94/26884; WO 94/24274; as well as U.S. Pat. Nos. 4,944,384 and 5,057,420 (which describe bovine nuclear transplantation), all of which are incorporated by reference in their entirety herein.
Nuclear transfer protocols typically include the steps of: (1) enucleating an oocyte; (2) isolating a cell to be combined with the enucleated oocyte; (3) inserting the cell, or nucleus isolated from the cell, into the enucleated oocyte to form a cybrid cell; (4) implanting the cybrid into the womb of the animal to form an embryo; and (5) allowing the embryo to develop.
Oocytes are typically isolated from either oviducts and/or ovaries of live animals, although they may be retrieved from deceased animals as well. Oocytes are typically matured in a variety of medium known to those of ordinary skill in the art prior to enucleation. Generally the oocytes used in nuclear transfer techniques are in the metaphase II cell-cycle stage. It is generally believed that oocytes are best fresh and non-preserved. Certain oocytes, such as cattle oocytes, are extremely sensitive to low temperatures and have not been found to be very useful after cryopreservation.
Enucleation of the oocyte can be performed in a number of manners, well known to those of ordinary skill in the art, including, aspiration (Smith & Wilmut, Biol. Reprod., 40: 1027–1035 (1989)), by use of DNA-specific fluorochromes (See, e.g., Tusnoda et al., J. Reprod. Fertil. 82: 173 (1988)), and irradiation with ultraviolet light (See, e.g., Gurdon, Q. J. Microsc. Soc., 101: 299–311 (1960)). Enucleation may also be effected by other methods known in the art, such as described in U.S. Pat. No. 4,994,384, herein incorporated by reference. Preferably, the oocyte is exposed to a medium containing a microfilament disrupting agent or tubulin-disrupting agent prior to and during, enucleation. Disruption of the microfilaments imparts relative fluidity to the cell membrane and underlying cortical cytoplasm such that a portion of the oocyte enclosed within the membrane can easily be aspirated into a pipette with minimal damage to cellular structures.
Until recently, donor nuclei have been conventionally isolated almost entirely from primordial germ cells and somatic embryo cells. During development certain genes are known to be altered in such a manner that they are no longer transcribed, so-called “imprinted”. Studies on imprinting have shown that “imprinting” is removed during germ cell formation (i.e. reprogramming).
It was not until the mid-1990's that reports of nuclear transfer form cultured cell lines arose. These reports (See, e.g., Wilmut et al., Nature (London) 385, 810–183) (1997)) suggest the usefulness of donor cells derived not only from embryos, but also, blastocysts, ovaries and other reproductive and sexually-related cells/tissues (e.g. the mammary epithelial cells, cumulus cells). Prior to the present invention, somatic cells derived from non-embryonic and non-reproductive/sexually related tissues (hereinafter referred to as, “NENS somatic cells”) were not found to be useful as donor cells in producing viable animal clones. In fact, as stated in U.S. Pat. No. 5,945,577 to Stice et al., until the late 1990s it was widely believed that only embryonic or undifferentiated cell types could direct any sort of fetal development in nuclear transfer techniques.
U.S. Pat. No. 5,945,577 to Stice et al., teaches advanced embryonic and fetal development from nuclear transfers from differentiated donor somatic cells to enucleated oocytes. U.S. Pat. No. 6,022,197 to Strelchenko et al., states that fibroblasts from a fibroblast cell culture derived from an adult ear punch may be used as nuclear donors in a nuclear transfer process. Both references, however, fail to demonstrate any viable animals being produced by their methodologies with NENS somatic cell nuclei donation.
With regard to somatic donor cells, prior to the present invention successful cloning experiments (that is, producing viable animal clones) have all entailed using donor nuclei from female donors. Researchers generally entertained the possibility that only female somatic cells associated with a reproductive organ(s) retain the capacity to be properly re-programmed by an oocyte environment (See, e.g., Capecchi, PNAS 97: 956–957 (Feb. 1, 2000)). That is, it was believed that male cells did not permit the proper execution of the complex changes in the patterns of genomic demethylation and methylation that normally accompany the process of early embryogenesis (which is necessary to maintain balanced growth between extra-embryonic and fetal tissues—See, e.g., Tilghman, S. M. Cell 96: 185–193 (1999)) due to an inherent incompatibility between male somatic nuclei and female oocyte cytoplasm (See, e.g., Capecchi, PNAS 97: 956–957 (Feb. 1, 2000)).
The miotic cell cycle is generally divided into four distinct phases: G1, S, G2, and M. The so-called “start event”, that is, the commitment to undergo another cell cycle, is made in the G1 phase. Once the “start event” has occurred, a cell passes through the remainder of the G1 phase which is a pre-DNA synthesis stage. The S phase which follows is when DNA synthesis takes place. The G2 phase is the period between DNA synthesis and mitosis. Mitosis occurs at the M phase. The donor nucleus (preferably in the G0 or G1 phase) is conventionally introduced into the recipient cells in the M phase of the cell cycle by either fusion or direct injection.
The donor cell is typically transferred into the perivitelline space of a enucleated oocyte to produce the cybrid. The recipient oocytes are conventionally arrested in the metaphase of the second meiotic division prior to fusion with the donor cell.
Fusion is typically induced by application of a DC electrical pulse across the contact/fusion plane, but additional AC current may be used to assist alignment of donor and recipient cells. Electrofusion produces a pulse of electricity that is sufficient to cause a transient breakdown of the plasma membrane and which is short enough that the membrane reforms rapidly. Fusion may also be induced by exposure of the cells to fusion-promoting chemicals, such as polyethylene glycol, or by way of an inactivated virus, such as the Sendai virus. In the case of small donor nuclei, microinjection directly into the oocyte may be preferred over fusion.
A cybrid is typically activated by electrical and/or non-electrical means before, during, and/or after fusion of the nuclear donor and recipient oocyte. Activation methods include electric pulses, chemically induced shock, penetration by sperm, increasing levels of divalent cations in the oocyte, and reducing phosphorylation of cellular proteins (as by way of kinase inhibitors) in the oocyte. The activated cybrids, or embryos, are typically cultured in medium well known to those of ordinary skill in the art, and include, without limitation, Tissue Culture Medium-199 (TCM-199)+10% fetal calf serum, Tyrodes-Albumin-Lactate-Pyruvate (TALP), Ham's F-10+10% fetal calf serum (FCS), synthetic oviductal fluid (“SOF”), B2, CR1aa, medium and high potassium simplex medium (“KSOM”).
The construction of embryos by nuclear transfer was first proposed by Spemann in the 1930s (Spemann, Embryonic Development and Induction 210–211, Hofner Publishing Co., New York (1938). It wasn't, however, until the early 1950s that it was demonstrated that nuclei could direct development (Briggs and King, Proc. Natl. Acad. Sci. USA 38 455–461 (1952)). The first successful nuclear transfer experiment using mammalian cells was reported by McGrath & Solter in 1983, wherein isolated pronuclei from a murine (mouse) zygote were inserted into an enucleated oocyte to result in live offspring (McGrath & Solter, Science 220: 1300–1312 (1983)).
One of first nuclear transfer experiments utilizing ovine embryonic cells as nuclear donors was reported by Willadsen in 1986 (Willadsen, Nature 320: 63–65 (1986)). Reports of cloning of animals using ovine embryonic cells as the nuclear donor occurred in the mid-1990s (Campbell et al., Nature 380: 64–66 (1996)); PCT Publication WO 95/20042). It was not, however, arguably not until 1997 that cloning of ovine animals became practicable using a technique described by Wilmut et al., (Wilmut et al., Nature 385: 810–183 (1997)). The Wilmut et al., publication describes a procedure which entails methodology for making the donor cell quiescent prior to the nuclear transfer. Ovine animals were produced using a mammary semantic cell donor and an enucleated oocyte.
The cloning of bovine animals using nuclear transfer techniques has not been reported to be as successful as in murine and ovine animals. Most reports have reported embryos that do not survive post utero. However, there are isolated reports of the successful cloning of cows (See, Kato et al., Science 282: 2095–2098 (1998); Wells et al., Biol. Reprod. 60: 996–1005 (1999); and Strelchenko et al., U.S. Pat. No. 6,011,197 (Issue Date: Jan. 4, 2000).
A major problem with all presently available nuclear transfer techniques is that they typically require donor cells which are relatively difficult to harvest and maintain, they require the use of relatively fresh donor cells or briefly cultured donor cells, cloning efficiency is low and they do not permit directed employment of genome manipulation techniques.
A large number of nuclear transfer studies have made use of embryonic cells or ovary cells as donor cells. The embryonic stem cell has been found to be a particularly useful cell as a donor cell in that it supports the development of enucleated oocytes to term. Genetic manipulation of mouse embryonic stem cells has revolutionized mouse genetic research. Unfortunately, embryonic stem cells are not readily available in other species.
The use of ungulate inner cell mass cells for nuclear transplantation has also been reported. Isolation of such cells tends to be cumbersome in particular given the need of these techniques for relatively fresh donor cells (which are available in low numbers) in order to make the cybrid. For example, embryonic cell lines used in a number of prior art nuclear transfer clonings were derived from embryos of less than 10 days gestation and were stored less than about 5 passages (See, e.g., Campbell et al., Nature, 380: 64–68 (1996); Stice et al., Biol. Reprod., 54: 100–110 (1996)). Such cells are also typically maintained on a feeder layer to prevent overt differentiation of the donor cell to be used in the cloning procedure. Because of the problems associated with harvesting such cells, a number of researchers have proposed using somatic cells as donor cells.
The major problem associated with somatic cell nuclear transfer has been a very low cloning efficiency. The efficiency of live births from somatic cell cloning using the method of cloning described by Wilmut et al., (Wilmut et al., Nature 385: 810–183 (1997)) has been estimated to be approximately 1 out of 300, that is, the cloning efficiency is at best 0.4% (i.e. number of cloned lambs divided by the number of nuclear transfers used to produce that number of cloned lambs). It is clear that the low cloning efficiency has significantly reduced commercialization prospects for such technology.
Successful somatic cell cloning has been largely limited to the use of donor cells that are either fresh (Wakayama et al., Nature (London) 394: 369–374 (1998)) or after short-term (under 10 passages) in vitro culture (Wilmut et al., Nature (London) 385: 810–813 (1997); Kato et al., Science 282: 2095–2098 (1998); Wells et al., Biol. Reprod. 60: 996–1005 (1997)); Schnieke et al., Science 278: 2130–2133 (1997); Cibelli et al., Science 280: 1256–1258 (1998)), which do not permit targeted gene manipulations, given the limitations of present technology.
There is a need, therefore, for a somatic cell nuclear transfer cloning technique which provides for the use of long-term cultured donor cells which retain the ability to produce cybrids capable of developing into viable animals, that provides for high cloning efficiency with easily harvested somatic donor cells, and that provides the opportunity to employ genetic manipulation techniques, in particular gene knock-out techniques, prior to formation of the cybrid.